Application of LYG1 in preparation of medicine for treating immune-related diseases

Abstract

The invention discloses an application of a cytokine LYG1 in preparation of a medicine for treating immune-related diseases. According to the present invention, the regulation effect and the mechanism of LYG1 in Tfh cells and cGVHD are deeply researched, such that the following important research results are obtained: the deletion of LYG1 inhibits the Tfh cell differentiation and the expression of characteristic molecules (BCL6, CXCR5, ICOS, TOX2 and the like), and the GC-B cell ratio and the antibody generation are reduced; the invention can be used for drugs and vaccines for autoimmune diseases such as cGVHD, immunodeficiency diseases and infectious diseases.

Description

Technical Field

[0001] The invention belongs to the field of biotechnology, and in particular to application of cytokine LYG1 in preparing medicines for treating immune-related diseases. Background Art

[0002] Tfh cells (Follicular helper T cells) are located in the B cell follicle area and are called follicular helper T cells. Since 2009, as three independent research groups reported internationally that Bcl6 is a key transcription factor required for Tfh cell differentiation, Tfh cells have gradually been accepted as an independent subpopulation that is different from other T cell subpopulations. Studies have shown that Tfh plays an important regulatory role in the formation of germinal centers (GCs). In the GC reaction, B cells undergo important physiological processes such as activation and proliferation, somatic high-frequency mutation, affinity maturation, antibody class switching, and memory B cell and plasma cell differentiation. These processes are inseparable from the participation of Tfh cells. If Tfh cells differentiate and function abnormally, a series of diseases will be caused. For example, if Tfh cells are defective in differentiation, GC formation will be inhibited, germinal center reactions will be weakened, antibody production will be reduced, and infectious diseases will be caused. On the contrary, if Tfh cells increase abnormally, a large number of autoantibodies will be produced, thereby causing autoimmune diseases. Although a large number of Tfh cell-related literature has been published in the past 10 years, and a variety of cytokines and transcription factors that regulate Tfh cell differentiation have been discovered, the regulatory mechanism of Tfh cell differentiation has not yet been fully elucidated. For example, it is still impossible to induce functional Tfh cells in vitro by cytokines, suggesting that there are still unknown cytokines that regulate Tfh cell differentiation. Therefore, further research on the regulatory mechanism of Tfh cell differentiation and the discovery of potential new cytokines that have regulatory effects on Tfh cells have important scientific theoretical significance and application value, which will help the prevention and treatment of autoimmune diseases, immunodeficiency diseases, infectious diseases, and the design and development of vaccines.

[0003] Tfh cell differentiation

[0004] The marker molecules on the surface of Tfh cells are CXCR5, PD1, ICOS, etc., and they mainly secrete functional cytokines IL-21. Current studies have shown that the differentiation of Tfh cells is a multi-stage, multi-factor-involved process. Figure 1 As shown, the initial CD4 + T cells recognize the MHC / antigen peptide complex presented by APCs such as DCs through TCR. At the same time, under the participation of ICOSL signals and IL-6 cytokines provided by DCs, CD4 + T cells begin to activate and express low levels of Bcl6 and CXCR5. Under the action of chemokines, this group of CD4+ T cells migrate to the TB cell junction, where they come into contact with antigen-specific B cells (providing TCR signals and costimulatory signals such as ICOS), further upregulating the expression of CXCR5 and Bcl6. Subsequently, Tfh precursor cells expressing CXCR5 continue to migrate to the B cell follicle area, interact with antigen-specific B cells (providing TCR signals and stimulatory signals such as ICOS and CD40), and induce the formation of germinal centers. In addition, the expression of Bcl6 inhibits the expression of Tbet, GATA3, and RORγt, thereby antagonizing the differentiation of Th1, Th2, and Th17 cells. After the above process, they eventually differentiate into mature Tfh cells.

[0005] Function of Tfh cells

[0006] The differentiation of Tfh cells depends on the signals provided by B cells, while the differentiation and function of germinal center B cells and plasma cells also depend on the assistance of Tfh cells. This complementary regulatory role is the core of the normal functioning of humoral immunity. First, in the process of B cell differentiation, only CD4 activated by continuous competitive contact with the same antigen can + T cells, B cells that obtain Tfh cell auxiliary signals can continue to survive, and then undergo division, clonal proliferation and somatic high-frequency mutation reactions. After multiple rounds of antigen-specific selection, GC B cells that finally express high-affinity antibodies leave the germinal center with the help of Tfh cells and differentiate into plasma cells or memory B cells. Secondly, Tfh cells can also regulate germinal center reactions by interacting with molecules such as ICOS and CD40L on their membrane surface and ICOSL and CD40 on the membrane surface of GC B cells. Finally, Tfh cells can also promote germinal center reactions by producing cytokines. IL-21 secreted by Tfh can promote B cell proliferation and differentiation and antibody production. GC B cells and plasma cells in IL-21 transgenic mice increased significantly, and antigen-specific IgG production by B cells in IL-21 knockout mice decreased. IFN-γ and IL-4 expressed by Tfh can regulate antibody class switching of GC B cells, such as inducing the production of IgG1, IgG2 or IgE.

[0007] Therefore, Tfh cells play a key role in GC reaction and formation (i.e., B cell activation and proliferation, somatic cell high-frequency mutation, affinity maturation, antibody class switching, and memory B cell and plasma cell differentiation).

[0008] Regulation of Tfh cell differentiation

[0009] The differentiation of Tfh cells is regulated by a variety of transcription factors, membrane molecules and cytokines. Bcl6 is a core transcription factor for Tfh cell differentiation and is highly expressed in Tfh cells. +In T cells, overexpression of Bcl6 can significantly promote Tfh cell differentiation and germinal center reaction; conversely, knocking out Bcl6 can block Tfh cell differentiation and germinal center reaction, but does not affect Th1, Th2 and Th17. In addition to the key transcription factor Bcl6, other transcription factors are also believed to promote Tfh cell differentiation, including Tox, Tox2, Ascl2, E2A, IRF4, c-Maf, Tcf7, Lef1, Stat3, etc. For example, Lef1 and Stat3 have an inducing effect on Bcl6 (5,16). The factors that negatively regulate Tfh cell differentiation mainly include Blimp1, Foxo1, Foxp1, Stat5 and Klf2. Among the many transcription factors, Blimp1 is the main negative regulatory factor. Blimp1 can directly inhibit Bcl6 expression, and overexpression of Blimp1 can significantly inhibit Tfh cell differentiation and germinal center reaction.

[0010] ICOS is another important molecule that regulates Tfh cell differentiation. ICOS is a co-stimulatory molecule of Tfh cells. In addition to regulating Tfh cell differentiation, it is also involved in the migration process of Tfh cells. The important regulatory effect of ICOS on Tfh cells has been found in multiple studies. Roquin can inhibit the expression of ICOS. In mice with both Roquin1 and Roquin2 deficiency, spontaneous Tfh cell differentiation and GC formation can be observed. In addition, the miR19-72 complex has also been shown to be involved in Tfh cell differentiation, and its role is mainly through the inhibitory molecules of PI3K, PHLPP2 and PTEN, which are also inhibitory molecules of the ICOS signaling pathway.

[0011] Cytokines are another important factor that regulates Tfh cell differentiation. Among the many cytokines, IL-6 is considered to be one of the most important cytokines. Studies have shown that in the early stages, Tfh cell differentiation is mainly regulated by IL-6, ICOS, IL-2 and TCR signals. In addition to TCR signals, IL-6 is considered to be the earliest signal to initiate Tfh cell differentiation. IL-6 induces newly activated initial CD4 +T cells transiently express Bcl6, which is a necessary condition for the early expression of CXCR5. Only T cells expressing CXCR5 can migrate to the junction of TB cells under the action of chemokines and further differentiate into mature Tfh cells. In IL-6-deficient mice, Tfh cells show early differentiation defects, but in most cases this defect can be compensated by IL-21 or IL-27. At the same time, some studies have found that IL-21-deficient mice do not show a significant decrease in the proportion and number of Tfh cells, but the formation of germinal centers is reduced. However, when IL-6 and IL-21 are knocked out in mice at the same time, a significant decrease in Tfh cells and a significant decrease in the production of antigen-specific antibodies are shown. IL-2 is the main negative regulatory molecule for Tfh differentiation. IL-2 can activate STAT5, thereby inducing the expression of Blimp1, achieving a negative regulatory effect on Tfh cell differentiation.

[0012] like Figure 2 As shown, in vitro, Th1, Th2, Th17 and iTreg cells can be generated from naive CD4 + T cells, stimulated by corresponding inducing cytokines, differentiate into Th cell subsets with consistent phenotypes and functions in vivo after several days of culture. However, it is still impossible to induce fully functional Tfh cells in vitro. It is only possible to induce Tfh-like cells that express Tfh characteristic genes but lack functionality. Current studies have shown that the addition of TGF-β and IL-12 or IL-23, IL-1β, and IL-6 in vitro can induce the differentiation of human Tfh-like cells and transiently express CXCR5, Bcl6, IL-21, and ICOS, but the same conditions cannot induce mouse Tfh cell differentiation. IL-12 can also induce human CD4 + T cells express IL-21, but cannot induce mouse expression. IL-6 can transiently induce mouse T cells to express Bcl6 and promote Tfh-like cell differentiation, but this phenomenon has not been found in human T cells. Anti-mouse IFN-γ, IL-4 and IL-2 antibodies and mouse IL-6 can induce mouse Tfh-like cells with high expression of genes such as CXCR5, Bcl6, PD1, and IL-21 in vitro. The above studies show that the regulatory mechanism of Tfh cell differentiation has not yet been fully clarified, and there are potential new cytokines and new mechanisms that affect Tfh cell differentiation that have not been discovered, which urgently need further research.

[0013] Tfh cells and chronic graft-versus-host disease

[0014] As mentioned above, Tfh cells can participate in the occurrence and development of various immune-related diseases, such as chronic graft-versus-host disease (cGVHD), by directly regulating GC formation and antibody production by B cells.

[0015] cGVHD is one of the most common complications after allogeneic hematopoietic stem cell transplantation, which seriously affects the quality of life of patients and lacks effective treatment measures. Unlike acute GVHD (mainly characterized by acute inflammatory response mediated by Th1 cells and proinflammatory cytokines), cGVHD is characterized by fibrotic changes similar to autoimmunity and multi-organ involvement, such as scleroderma, bronchiolitis obliterans, and liver and intestinal fibrosis. The treatment effect of patients with hormone-dependent or refractory cGVHD is poor. Currently, only ibrutinib (targeting the B cell BTK pathway) has been approved by the US Food and Drug Administration (FDA) for the treatment of cGVHD.

[0016] In terms of pathogenesis, cGVHD is often driven by germinal center reactions, and Tfh cells interact with GC B cells to produce cGVHD-causing antibodies. Stimulating antibodies against platelet-derived growth factor receptor (PDGFR) can be detected in cGVHD patients, and higher levels are detected in cGVHD patients with systemic skin involvement and / or pulmonary fibrosis, but not in patients who do not develop cGVHD. Studies in mouse allogeneic hematopoietic stem cell transplantation models have found that the onset of cGVHD depends on Tfh cells. Knocking out CXCR5 or neutralizing IL-21, CD40L, and ICOS can effectively attenuate allogeneic antibody responses and improve cGVHD in the lungs. Neutralizing IL-21 with neutralizing antibodies can alleviate the symptoms of mouse skin cGVHD. Knocking out Bcl6 in donor T cells reduces the ratio of Tfh cells to GC B cells in the spleen of recipient mice, significantly reduces antibody and collagen deposition in the lungs, and attenuates cGVHD in the lungs. CD4 + C57BL / 6 mice with T cell-specific knockout of MiR-17-92 were used as donors, and BALB / c mice were used as recipients to construct a cGVHD scleroderma model. The study found that the differentiation of donor-derived Tfh cells was blocked, the production of GC B cells was reduced, the collagen deposition and antibody deposition in the recipient mouse skin were reduced, and the incidence of cGVHD was weakened. In addition, in the lupus-like cGVHD model, blocking MiR-17-92 in the recipient mouse can reduce the occurrence of proteinuria. Summary of the invention

[0017] The purpose of the present invention is to provide the use of cytokine LYG1 in the preparation of drugs for treating immune-related diseases.

[0018] According to a first aspect of the present invention, there is provided a use of cytokine LYG1 in the preparation of a medicament for treating autoimmune-related diseases.

[0019] Preferably, the autoimmune-related diseases include but are not limited to graft-versus-host disease, autoimmune diseases such as systemic lupus erythematosus, and kidney-related diseases.

[0020] Preferably, the active ingredient of the drug is a LYG1 inhibitor or neutralizing antibody or a related drug that inhibits LYG1 expression.

[0021] Preferably, the active ingredient of the drug can inhibit Tfh cell development and differentiation or antibody production.

[0022] According to a second aspect of the present invention, there is provided a method for preparing a drug for treating diseases related to immune deficiency or hypofunction.

[0023] Preferably, the related diseases caused by the immune deficiency or dysfunction are bacterial infection and viral infection.

[0024] Preferably, the active ingredient of the drug is LYG1, and the LYG1 is used to promote the development and differentiation of Tfh cells or the production of antibodies.

[0025] According to the third aspect of the present invention, the present invention provides the use of cytokine LYG1 in the preparation of a vaccine for promoting the development and differentiation of Tfh cells or the production of antibodies.

[0026] Preferably, the LYG1 is an auxiliary drug or adjuvant of a vaccine.

[0027] The beneficial technical effect of the present invention is that LYG1 deficiency is used to inhibit Tfh cell differentiation and the expression of characteristic molecules (BCL6, CXCR5, ICOS, TOX2, etc.), thereby reducing the proportion of GC B cells and antibody production. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] Figure 1 It is a schematic diagram of the Tfh cell division process;

[0029] Figure 2 This is a schematic diagram of cytokines that induce Th cell division in vitro;

[0030] Figure 3 It is a schematic diagram of the present invention confirming through experiments that LYG1 deficiency can improve cGVHD in mice;

[0031] Figure 4 It is a schematic diagram of the present invention confirming through experiments that LYG1 deficiency improves cGVHD and is related to Tfh;

[0032] Figure 5It is a schematic diagram of the present invention confirming through experiments that LYG1 deficiency inhibits the differentiation of Tfh cells;

[0033] Figure 6 It is a schematic diagram of the present invention confirming through experiments that LYG1 deficiency inhibits Tfh cell function;

[0034] Figure 7 It is a schematic diagram of the present invention confirming through experiments that LYG1 deficiency inhibits Tfh differentiation independently of IL-6;

[0035] Figure 8 It is a schematic diagram of the results of culturing naive CD4+ T cells from LYG1- / - mice or their WT littermates under Tfh polarization conditions in the presence of rhLYG1 (0, 20 or 200 ng / mL);

[0036] Fig. 9 Graph showing the results of CD4+ T cells incubated with IL-6 (20 ng / mL), rhLYG1 (20 or 200 ng / mL) or buffer control for 10 minutes. DETAILED DESCRIPTION

[0037] The inventors used immunogenomics technology to carry out bioinformatics selection, functional screening and systematic functional research of human potential new cytokine encoding genes, in order to discover new cytokines that regulate immune responses. LYG1 (lysozyme G-like 1) is one of the potential new cytokines screened using this platform.

[0038] First, the study found that LYG1 has the following characteristics: 1) It is a classic secretory protein with a small molecular weight and a mature form of about 20 kDa; 2) Although bioinformatics suggests that LYG1 belongs to the G-type lysozyme family, it has no lytic activity; 3) It is expressed in CD4 + In T cells, the effect of LYG1 recombinant protein has a clear dose-dependent relationship, which conforms to the bell-shaped curve. Its optimal dose is about 10ng / ml, and its effect is highly effective. The above characteristics are consistent with the action characteristics of cytokines, that is, cytokines function by binding to corresponding receptors, usually with dose dynamics, and too high or too low concentrations cannot achieve the best effect. This suggests that LYG1 is a CD4 + A potential new cytokine with immunomodulatory effects on T cells.

[0039] The present inventors have conducted in-depth and systematic research on the role and mechanism of LYG1 in acute GVHD (acute GVHD, aGVHD). - / - Mouse T cells can significantly improve aGVHD in recipient mice, and Lyg1 - / -The proportion of Treg cells in mice increased. The inventors detected the peripheral blood of patients with aGVHD who received allogeneic hematopoietic stem cell transplantation and found that compared with patients with 0-1 degree aGVHD, the level of LYG1 in the serum of patients with 2-4 degree aGVHD was significantly increased, suggesting that LYG1 has a clinical correlation with the severity of aGVHD. This part of the work has been submitted to the mainstream journal Biology of Blood and Marrow Transplantation (under review) in the field of hematology and selected as a poster presentation at the EBMT conference (European Blood and Marrow Transplantation Annual Meeting).

[0040] In addition, preliminary studies have found that Lyg1 - / - The proportion of Tfh cells in mice was significantly reduced, the expression of ICOS in Tfh cells was significantly reduced, and the proportion of GC B cells and antibody production were significantly reduced. - / - Mouse T cells can significantly improve cGVHD in recipient mice. The above research results all suggest that LYG1 is a new molecule that promotes Tfh cell differentiation. In order to obtain the mechanism of action of LYG1 in promoting Tfh cell differentiation, the inventors used LYG1 recombinant protein and Lyg1 - / - In mice, the role and mechanism of the potential new cytokine LYG1 in Tfh cell differentiation in vitro, antigen-specific differentiation in vivo and mouse cGVHD model were systematically studied in combination with clinical specimen analysis. The implementation of the present invention will further deepen the understanding of Tfh cells and discover a new, previously unreported molecule that regulates Tfh cell differentiation, which will provide new strategies for the prevention and treatment of related immune diseases and vaccine design, and has important scientific theoretical significance and application value.

[0041] Research results of the present invention

[0042] 1. LYG1 participates in the development of cGVHD by regulating Tfh cell differentiation

[0043] Wild-type (Lyg1+ / +) mice and Lyg1 knockout (Lyg1- / -) mice were used as donor mice to construct cGVHD models, and the body weight, cGVHD clinical phenotype, and survival of each group of model mice were dynamically monitored. Compared with the Lyg1+ / + group of mice, the survival rate of the Lyg1- / - group of mice was significantly increased ( Figure 3 A), slow weight loss ( Figure 3 B), clinical cGVHD score decreased ( Figure 3 C), liver, lung and colon damage was significantly alleviated, lymphocyte infiltration was reduced, and histological injury scores were significantly reduced ( Figure 3D), collagen deposition in liver and lungs was significantly reduced ( Figure 3 E), confirmed that LYG1 deficiency can improve cGVHD in mice ( Figure 3 ).

[0044] Through further research, the inventors found that in this mouse cGVHD model, the IgG immune complex deposition in the colon, lung and skin of the Lyg1- / - group mice was significantly reduced compared with that of the Lyg1+ / + group mice ( Figure 4 A), lymph node germinal centers are significantly reduced ( Figure 4 B), the proportion of Tfh and GCB cells decreased ( Figure 4 C), indicating that LYG1 deficiency ameliorates cGVHD and is associated with Tfh.

[0045] The representative results of this study confirmed that LYG1 participates in the occurrence and development of cGVHD by regulating the differentiation of Tfh cells, and clarified the role and mechanism of LYG1 in cGVHD. This is an important supplement to the research on the pathogenesis of Tfh cells in cGVHD, and also provides a potential new treatment method for the prevention and treatment of cGVHD.

[0046] The cGVHD mouse model was established, and the phenotype of the mice was observed within 60 days after transplantation. On day 0, BALB / c mice were irradiated with a lethal dose of 800 cGy, and spleen cells (1.25×10 6 ) and bone marrow cells (2.5×10 6 ). Mice in the bone marrow group (control) were only given BM infusion. A, survival status, B, mouse body weight changes, C, cGVHD score. D, histological changes in liver, lung and colon (HE staining). Marking = 200μm. E, collagen deposition in liver, lung and colon (Masson staining). Blue indicates collagen deposition. Marking = 200μm. AC, n = 10 (number of mice in each group), DE, n = 5, * is P < 0.05, ** is P < 0.

[0047] like Figure 4 As shown, in Figure 3 In the model, on the 35th day after transplantation, A, the colon and lung antibody deposition in the two groups of models was detected (IgG immunofluorescence staining). Green fluorescence indicates antibody deposition. B, the formation of lymph node germinal centers in the two groups of models was detected (PNA immunofluorescence staining). Green fluorescence indicates germinal centers. The right figure is a statistical chart. n=5, **P<0.01. C, the proportion of lymph node Tfh and GCB cells in the two groups of models was detected (flow cytometry method). Tfh cells are CD4+PD1+CXCR5+, and GCB cells are CD19+Fas+GL7+. n=3.

[0048] Representative Results 2 LYG1 promotes Tfh cell differentiation, germinal center B cells and antibody production.

[0049] First, the inventors found that the percentage of Tfh cells in the spleen and lymph nodes (LN) of Lyg1 KO mice was significantly reduced compared with that of WT littermates without any treatment ( Figure 5 A). To investigate the role of Lyg1 in Tfh cells, the inventors immunized Lyg1 KO mice and WT littermates with KLH. Interestingly, there was no significant difference in the percentage and absolute number of CD4+ T cells in the draining lymph nodes (DLN) between Lyg1 KO mice and their WT littermates ( Figure 5 B). However, after KLH immunization, the percentage of Tfh cells (PD1+CXCR5+) within the CD4+ T cell population was significantly decreased in Lyg1 KO mice compared with WT mice ( Figure 5 C). BCL6 is a specific transcription factor for Tfh cells and is essential for their differentiation. Notably, the expression of BCL6 (BCL6+CXCR5+) in CD4+ T cells in Lyg1 KO mice was significantly reduced compared with WT mice ( Figure 5 D). Since ICOS and IL-21 play a key role in Tfh cell differentiation, the inventors further evaluated their expression levels in Tfh cells. The inventors' results showed that the expression of ICOS and IL-21 was significantly reduced in Tfh cells of Lyg1 KO mice ( Figure 5 E and Figure 5 F). To explore the effect of Lyg1 on antigen-specific Tfh cells, the inventors used Lyg1 KO / OT-II mice and immunized them and their WT littermates with OVA. Consistent with the results of KLH immunization, the percentage of Tfh cells within the CD4+ T cell population in LN was significantly reduced in Lyg1 KO mice compared with WT mice after OVA immunization ( Figure 5 G). Collectively, these findings suggest that LYG1 loss inhibits Tfh cell differentiation.

[0050] LYG1 deficiency inhibits Tfh cell differentiation, so what is the effect on Tfh cell function? Tfh cells play an important role in promoting the production of germinal center (GC) B cells and antibody production. First, the percentage of GC B cells in the lymph nodes and spleen of Lyg1KO mice was significantly reduced compared with that of WT littermates without any treatment ( Figure 2 A). In addition, the inventors examined the differentiation of GC B cells and found that KLH ( Figure 2 B) or OVA( Figure 2C) After stimulation, the proportion of GC B cells within the CD19+B cell population in Lyg1 KO mice was significantly lower than that in WT mice in LN. These findings suggest that LYG1 knockout inhibits the differentiation of GC B cells. In addition, the inventors also analyzed antibody production. The serum immunoglobulin levels of Lyg1 KO mice were lower than those of WT mice ( Figure 2 D). Is the inhibitory effect of GC B cells and antibody production in LYG1 knockout mice caused directly by LYG1 knockout or indirectly by the impaired Tfh differentiation caused by LYG1 deficiency? To investigate this, we sorted Tfh cells from WT and Lyg1 KO mice immunized with NP-KLH on day 7 and co-cultured them with B cells from untreated WT and Lyg1 KO mice for 6 days. Figure 2 E shows that among the four groups, immunoglobulin production was reduced in the Lyg1 KO Tfh cell and WT B cell groups compared with the co-cultured WT Tfh and WT B cell groups. In addition, Tfh cells from WT mice could restore immunoglobulin production in Lyg1 KO mice compared with the co-cultured Lyg1 KO Tfh and Lyg1 KO B cell groups.

[0051] Representative results 2 and 3 indicate that LYG1 may be another important factor regulating the differentiation and development of Tfh cells, and also provide new ideas for the treatment of antigen-antibody deposition-related diseases and vaccine design.

[0052] Figure 5 A shows that in the absence of any treatment, Tfh cell differentiation was attenuated in the spleen and lymph nodes (LNs) of LYG1 KO mice. Flow cytometry staining was used to analyze the proportion of Tfh cells (PD1+CXCR5+) in CD4+ T cells in WT and LYG1 KO mice. BF, WT and LYG1 KO mice were immunized with NP-KLH, and their draining lymph nodes (DLN) were removed on day 7 to obtain lymphocytes. Figure 5 B shows the proportion and absolute number of CD4+ T cells in DLN. Figure 5 C Left panel shows representative flow cytometry images of PD1+CXCR5+ cells. Figure 5 C right panel shows the proportion of Tfh cells (PD1+CXCR5+) among CD4+T cells. Figure 5 D Left panel shows representative flow cytometry images of BCL6+CXCR5+ cells. Figure 5 D The right panel shows the proportion of Tfh cells (BCL6+CXCR5+) among CD4+ T cells. Figure 5 E The left panel shows the flow cytometry histogram of ICOS expression in Tfh cells, and the right panel shows the mean fluorescence intensity of ICOS expression. Figure 5F shows that the expression of IL21 mRNA was detected by qPCR after sorting DLN CD4+T cells. Figure 5 G shows the ratio of Tfh cells in CD4+ T cells in DLN at day 7 after OVA immunization in WT / OT-II and LYG1 KO / OT-II mice. The experiment was repeated 2-3 times, and representative results are shown in the figure. The number of mice in each group was equal to or greater than 5. Statistical results are expressed as mean ± SD, *p < 0.05, **p < 0.01, ***p < 0.001.

[0053] Figure 6 It was shown that LYG1 deficiency inhibited Tfh cell function. Figure 6 A shows that the number of GCB cells in the spleen and lymph nodes (LNs) of LYG1KO was reduced in the absence of any treatment. Figure 6 B shows the proportion of GCB cells (GL7+CD95+) in CD19+B cells of WT and LYG1KO mice analyzed by flow cytometry. WT and LYG1KO mice were immunized with NP-KLH, and their draining lymph nodes (DLN) were removed on day 7 to obtain lymphocytes. Figure 6 B Left panel shows representative flow cytometry images of GL7+CD95+ cells. Figure 6 B Right panel shows the ratio of GCB cells (GL7+CD95+) among CD19+B cells. Figure 6 C shows the ratio of CD19+ B cells to GCB cells in DLN on day 7 after OVA immunization of WT / OT-II and Lyg1 KO / OT-II mice. Serum immunoglobulin levels in WT and LYG1 KO mice were measured by flow cytometry on day 7 after KLH immunization. LYG1 deficiency reduces the ability of Tfh cells to stimulate B cell antibody production. WT mice and LYG1 KO mice were immunized with NP-KLH. On day 7, Tfh cells (CD4+PD1+CXCR5+) in DLN were sorted and co-cultured with B cells from WT mice and LYG1 KO mice for 6 days. Enzyme-linked immunosorbent assay (ELISA) was used to detect the level of antibody production in the culture supernatant. The vertical axis in the figure represents the absorbance value of OD450. The experiment was repeated 2-3 times, and representative results are shown in the figure. The number of mice in each group was equal to or greater than 4. Statistical results are expressed as mean ± SD, *p < 0.05, **p < 0.01, ***p < 0.001.

[0054] Representative Results (Mechanism Exploration) 3. LYG1 may promote Tfh differentiation through a mechanism independent of IL-6.

[0055] 3.1 In the in vivo Tfh cell differentiation model, LYG1 deficiency inhibited Tfh cell differentiation independently of IL-6.

[0056] IL-6 is a key cytokine that induces Tfh differentiation. In order to study whether LYG1 regulates Tfh differentiation through IL-6, the inventors used antibodies against IL-6 or IL-6 receptor (IL-6R) to block IL-6 signaling. LYG1- / - mice and control WT mice received intraperitoneal injections of isotype control antibodies, IL-6 neutralizing antibodies, IL-6R neutralizing antibodies, or a mixture of IL-6 and IL-6R neutralizing antibodies, and then the mice were immunized with NP-KLH. Seven days after immunization, flow cytometric analysis of BCL6, PD1, CXCR5, and ICOS expression was performed on inguinal lymph node (ILN) lymphocytes. The results showed that blocking IL-6 signals with neutralizing antibodies reduced the proportion of BCL6+ and ICOS+ cells in CD4+T cells of WT mice, and reduced the absolute counts of CD4+PD1+CXCR5+Tfh cells (see Figure 7 A- Figure 7 C). Notably, LYG1 deficiency further reduced the proportion of BCL6+ and ICOS+ cells and absolute Tfh cell counts in IL-6-neutralized mice, suggesting that LYG1 inhibits Tfh differentiation through an IL-6-independent mechanism.

[0057] Figure 7 LYG1 deficiency inhibits Tfh differentiation independently of IL-6. LYG1- / - mice and their control WT mice received intraperitoneal injections of isotype control antibodies (WT, KO), IL-6 neutralizing antibodies (WT-IL6 block, KO-IL6 block), IL-6R neutralizing antibodies (WT-IL6R block, KO-IL6R block), or a mixture of IL-6 and IL-6R neutralizing antibodies (WT-IL6 IL6R block, KO-IL6IL6R block). After antibody administration, mice were immunized with NP-KLH. Seven days after immunization, the expression of BCL6, CXCR5, PD1, and ICOS on ILN lymphocytes was analyzed by flow cytometry. Figure 7 A shows the ratio of CD4+ T cells to BCL6+ cells. Figure 7 B shows the absolute counts of CD4+PD1+CXCR5+Tfh cells. Figure 7 C shows the proportion of CD4+ T cells and ICOS+ cells. N≥4, *p<0.05, **p<0.01, ***p<0.001.

[0058] 3.2 In the in vitro Tfh-like cell differentiation model, rhLYG1 promoted Tfh differentiation independently of IL-6 and could restore the inhibitory effect of LYG1 deficiency on Tfh cell differentiation.

[0059] BCL6 and TOX2 are transcription factors that drive Tfh differentiation. In order to further study the regulatory function of LYG1 in Tfh differentiation, the inventors used BCL6 and TOX2 as markers to evaluate the effect of rhLYG1 on inducing the differentiation of naive CD4+T cells into Tfh-like cells in vitro. In the absence or presence of rhLYG1, naive CD4+T cells from LYG1- / - mice or their control WT mice were cultured alone or in combination with IL-6 neutralizing antibodies under Tfh-like in vitro polarization conditions for 3 days. qPCR analysis showed that both LYG1 knockout and IL-6 neutralization reduced the expression of BCL6 and TOX2, respectively. After the addition of IL-6 neutralizing antibodies to the LYG1 knockout group, the expression of BCL6 and TOX2 was the lowest ( Figure 8 A, Figure 8 B). More importantly, rhLYG1 restored the down-regulated BCL6 and TOX2 ( Figure 8 A, Figure 8 B).

[0060] Figure 8 Naive CD4+ T cells from LYG1- / - mice or their WT littermates were cultured under Tfh polarizing conditions in the presence of rhLYG1 (0, 20, or 200 ng / mL), alone or in combination with IL-6 neutralizing antibody (IL-6block) for 3 days. BCL6 (A) and TOX2 (B) mRNA levels were detected by qPCR. N≥4, *p<0.05, **p<0.01, ***p<0.001.

[0061] 3.3LYG1 promotes Tfh differentiation not through the IL6-STAT3 signaling pathway

[0062] Studies have reported that IL-6 induces BCL6-mediated Tfh differentiation through STAT3. The inventors used flow cytometry to detect the level of phosphorylated STAT3 (p-STAT3) in CD4+ T cells. Figure 3 As shown in A, IL-6 induced STAT3 phosphorylation in CD4+T lymphocytes, while rhLYG1 showed no significant effect. These results support that LYG1 promotes Tfh differentiation through a mechanism independent of IL-6-STAT3 signaling. Studies have reported that ICOS drives BCL6-mediated Tfh differentiation through PI3K-AKT. In this study, the inventors performed transcriptome sequencing on LYG1- / - and WT CD4+T cells, and KEGG analysis showed that the PI3K-AKT pathway was downregulated in LYG1- / -CD4+T cells ( Fig. 9 B), suggesting that LYG1 may promote Tfh differentiation by enhancing ICOS / PI3K-AKT signaling, which needs further verification.

[0063] Fig. 9 A shows that CD4+ T cells were incubated with IL-6 (20 ng / mL), rhLYG1 (20 or 200 ng / mL) or buffer control for 10 minutes. The expression level of p-STAT3 in CD4+ cells was detected by flow cytometry. N=4, *p<0.05, ns=not significant. Fig. 9 B shows transcriptome sequencing (KEGG) analysis of LYG1- / - and WT CD4+ T cells.

[0064] In summary, LYG1 deficiency also reduced Tfh differentiation in vitro and in vivo when IL-6 signaling was blocked by neutralizing antibodies, and rhLYG1 was able to restore the effects of IL-6 neutralizing antibodies or LYG1 deficiency that inhibited Tfh differentiation. These results suggest that LYG1 may promote Tfh differentiation through a mechanism independent of IL-6.

[0065] Based on the above research, the present invention provides the following applications in treating or preventing autoimmune related diseases:

[0066] A cytokine LYG1 is used in the preparation of a drug for treating autoimmune-related diseases.

[0067] The autoimmune-related diseases include but are not limited to graft-versus-host disease, autoimmune diseases such as systemic lupus erythematosus, and kidney-related diseases.

[0068] The active ingredient of the drug is a LYG1 inhibitor or neutralizing antibody or a related drug that inhibits LYG1 expression.

[0069] The active ingredients of the drug can inhibit the development and differentiation of Tfh cells or the production of antibodies.

[0070] A cytokine LYG1 is used in the preparation of a drug for treating related diseases caused by immune deficiency or dysfunction.

[0071] Related diseases caused by immune deficiency or dysfunction include bacterial infection and viral infection.

[0072] The active ingredient of the drug is LYG1, which is used to promote Tfh cell development and differentiation or antibody production.

[0073] Application of cytokine LYG1 in the preparation of vaccines for promoting Tfh cell development and differentiation or antibody production.

[0074] LYG1 is an auxiliary drug or adjuvant for vaccines.

[0075] Although the present invention is described in detail above, the present invention is not limited thereto, and those skilled in the art can make various modifications based on the principles of the present invention. Therefore, all modifications made based on the principles of the present invention should be understood to fall within the protection scope of the present invention.

Claims

1. Application of cytokine LYG1 in the preparation of drugs for treating autoimmune related diseases.

2. The use according to claim 1, characterized in that: The autoimmune-related diseases include but are not limited to graft-versus-host disease, autoimmune diseases, and kidney-related diseases.

3. The use according to claim 2, characterized in that: The active ingredient of the drug is a LYG1 inhibitor or neutralizing antibody or a related drug that inhibits LYG1 expression.

4. The use according to claim 3, characterized in that: The active ingredients of the drug can inhibit the development and differentiation of Tfh cells or the production of antibodies.

5. The cytokine LYG1 is used in the preparation of drugs for treating related diseases caused by immunodeficiency or dysfunction.

6. The use according to claim 4, characterized in that: The related diseases caused by the immune deficiency or dysfunction are bacterial infection and viral infection.

7. The use according to claim 5, characterized in that: The active ingredient of the drug is LYG1.

8. The use according to claim 7, characterized in that: The active ingredients of the drug can promote the development and differentiation of Tfh cells or the production of antibodies.

9. Application of cytokine LYG1 in the preparation of vaccines that can promote Tfh cell development and differentiation or antibody production.

10. The use according to claim 9, characterized in that: The LYG1 is an auxiliary drug or adjuvant of the vaccine.

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